Microglial activation resulting from CD40-CD40L interaction after beta-amyloid stimulation.

Alzheimer's disease (AD) has a substantial inflammatory component, and activated microglia may play a central role in neuronal degeneration. CD40 expression was increased on cultured microglia treated with freshly solublized amyloid-beta (Abeta, 500 nanomolar) and on microglia from a transgenic murine model of AD (Tg APPsw). Increased tumor necrosis factor alpha production and induction of neuronal injury occurred when Abeta-stimulated microglia were treated with CD40 ligand (CD40L). Microglia from Tg APPsw mice deficient for CD40L demonstrated reduction in activation, suggesting that the CD40-CD40L interaction is necessary for Abeta-induced microglial activation. Finally, abnormal tau phosphorylation was reduced in Tg APPsw animals deficient for CD40L, suggesting that the CD40-CD40L interaction is an early event in AD pathogenesis.

SOLUBLE CD40 LIGAND IN DEMENTIA.

Some 15-20% of the population over the age of 65 years suffer from dementia, currently one of the leading causes of death behind cardiovascular diseases, cancer and cerebrovascular diseases. The major forms of dementia share in common overactivation of the CD40-CD40-L complex, leading to high levels of proinflammatory cytokine production by immune cells of the central nervous system (CNS), including microglia and astrocytes. Consequently, both neuronal survival and signaling are negatively affected, leading to the characteristic progressive loss of higher cortical functions. We have reviewed the literature concerning the involvement of this complex in the pathology of three major forms of dementia: Alzheimer's-type, HIV-associated and vascular dementia. This is followed by a discussion of current preclinical and clinical therapies that may influence this interaction, and thus point the way toward a future neuroimmunological approach to inhibiting the effects of CD40-CD40-L in neuropsychiatric disease.

CD45 opposes beta-amyloid peptide-induced microglial activation via inhibition of p44/42 mitogen-activated protein kinase.

Reactive microglia have been suggested to play a role in the Alzheimer's disease (AD) process, and previous studies have shown that expression of CD45, a membrane-bound protein-tyrosine phosphatase (PTP), is elevated in microglia in AD brain compared with controls. To investigate the possible role of CD45 in microglial responsiveness to beta-amyloid (Abeta) peptides, we first co-treated primary cultured microglia with a tyrosine phosphatase inhibitor [potassium bisperoxo (1,10-phenanthroline) oxovanadate (phen), 5 micrometer] and freshly solubilized Abeta peptides (1000 nm). Data show synergistic induction of microglial activation as evidenced by tumor necrosis factor alpha (TNF-alpha) production and nitric oxide (NO) release, both of which we show to be dependent on activation of p44/42 mitogen-activated protein kinase (MAPK). Furthermore, co-treatment with phen and Abeta peptides results in microglia-induced neuronal cell injury. Stimulation of microglial CD45 by anti-CD45 antibody markedly inhibits these effects via inhibition of p44/42 MAPK, suggesting that CD45 is a negative regulator of microglial activation. Accordingly, primary cultured microglia from CD45-deficient mice demonstrate hyper-responsiveness to Abeta, as evidenced by TNF-alpha release, NO production, and neuronal injury after stimulation with Abeta peptides. As a validation of these findings in vivo, brains from a transgenic mouse model of AD [transgenic Swedish APP-overexpressing (Tg APP(sw)) mice] deficient for CD45 demonstrate markedly increased production of TNF-alpha compared with Tg APP(sw) mice. Taken together, these results suggest that therapeutic agents that stimulate the CD45 PTP signaling pathway may be effective in suppressing microglial activation associated with AD.

Cholesterol accumulates in senile plaques of Alzheimer disease patients and in transgenic APP(SW) mice.

Mounting evidence suggests that cholesterol may contribute to the pathogenesis of Alzheimer disease (AD). We examined whether cholesterol might be present in senile plaques, a hallmark neuropathological feature of AD. We employed 2 different fluorometric-staining techniques (filipin staining and an enzymatic technique) for the determination of cholesterol in brains of postmortem confirmed AD patients and in nondemented, age-matched histopathologically normal controls. AD patient brains showed abnormal accumulation of cholesterol in congophilic/birefringent dense cores of senile plaques that was essentially absent in histopathologically normal controls. To determine whether increased senile plaque-associated cholesterol occurred generally in all plaques or was restricted to a specific subset, quantitative analysis was performed. Data indicate abnormal accumulation of cholesterol in cores of mature plaques but not in diffuse or immature plaques. Additionally, transgenic mice that overexpress the "Swedish" amyloid precursor protein (Tg APP(SW), line 2576) exhibited a similar pattern of abnormal cholesterol accumulation in mature, congophilic amyloid plaques at 24 months of age that was absent in their control littermates or in 8-month-old Tg APP(SW) mice (an age prior to amyloid deposition). Taken together, our results imply a link between cholesterol and AD pathogenesis and suggest that cholesterol plays an important role in the formation and/or progression of senile plaques.

Intracisternal increase of superoxide anion production in a canine subarachnoid hemorrhage model.

BACKGROUND AND PURPOSE: Reactive oxygen species (ROS) are thought to be primary in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH). However, as direct evidence of ROS has not yet been demonstrated in cerebral vasospasm, we sought to substantiate superoxide anion (.O(2)(-)) generation in the subarachnoid space after SAH using a modification of Karnovsky's manganese/diaminobenzidine (Mn(2+)/DAB) technique. METHODS: SAH or sham operation was induced according to a 2-hemorrhage model in a total of 24 beagle dogs. On day 2 or 7 after SAH or sham operation, dogs were intrathecally infused with buffer containing Mn(2+) and DAB, and the brain stem was prepared for light and electron microscopy. Possible colocalization of ferrous (Fe(2+)) or ferric (Fe(3+)) iron ions with.O(2)(-) was also examined with the use of Turnbull blue or Berlin blue staining, respectively. RESULTS: Light microscopy revealed amorphous, amber deposits within the subarachnoid hematoma, the periarterial space, and the tunica adventitia of the basilar artery on days 2 and 7 after SAH.O(2)(-) deposits were eliminated by addition of superoxide dismutase or exclusion of either Mn(2+) or DAB from the perfusate, confirming the specificity of the reaction. These deposits were colocalized with blue reaction deposits indicating Fe(2+) and Fe(3+). Within the subarachnoid space,.O(2)(-) indicating electron-dense fine granules were preferentially located around degenerated erythrocytes and, secondarily, infiltrating macrophages and neutrophils. CONCLUSIONS: We show direct evidence for enhanced production of.O(2)(-) and Fe(2+)/Fe(3+) iron ions in the subarachnoid space after SAH, lending further support to the pathogenic role of ROS in cerebral vasospasm after SAH.

Soluble beta-amyloid peptides mediate vasoactivity via activation of a pro-inflammatory pathway.

Freshly solubilized beta-amyloid (Abeta) peptides display vasoactive properties, increasing both the magnitude and the duration of endothelin-1-induced vasoconstriction. We show that Abeta vasoactivity is mediated by the stimulation of a pro-inflammatory pathway involving activation of secretory phospholipase A(2) (PLA(2)), mitogen activated protein kinase (MAPK) kinase (MEK1/2), p38 MAPK, cytosolic PLA(2), and the release of arachidonic acid. Ultimately, arachidonic acid is metabolized into proinflammatory eicosanoids via the 5-lipoxygenase and cyclooxygenase-2 (COX-2) enzymes, both of which we show to be required for A beta vasoactivity. Accordingly, p38 MAPK activity is higher in the brains of transgenic mice that overproduce A beta, and COX-2 immunoreactivity is increased in the cerebrovasculature of these transgenic animals. Taken together, our data show that freshly solubilized A beta peptides can trigger a pro-inflammatory reaction in the vasculature that can be blocked by inhibiting specific target molecules, providing the basis for novel therapeutic intervention.

Activation of microglial cells by the CD40 pathway: relevance to multiple sclerosis.

It is well known that microglial cells perform a key role in mediating inflammatory processes, which are associated with neurodegenerative diseases such as multiple sclerosis (MS). In this study, we report that CD40 expression on microglia is greatly enhanced by a low dose (10 U/ml) of IFN-gamma. We also find that ligation of microglial CD40 by CD40L triggers a significant production of TNF-alpha. Activation of microglia by ligation of CD40 in the presence of IFN-gamma results in cultured cortical neuronal injury, which is markedly attenuated by blockade of the CD40 pathway or neutralization of TNF-alpha. Finally, we find significant levels of IFN-gamma and TNF-alpha in the medium of co-cultured activated CD4+ T cells and microglial cells, showing that microglia can supply the CD40 receptor to activated CD4+ T cells and suggesting that this cellular interaction is a key event in MS pathophysiology.

Association of a functional mu-opioid receptor allele (+118A) with alcohol dependency.

Genetic association studies have implicated the TaqI A1 allele of the human dopamine D2 receptor gene (DRD2) as a risk-determining factor for alcohol dependency. However, as alcoholism is a disease of polygenic inheritance, the percentage of overall disease variance explained by the TaqI A1 allele is small. In searching for other genetic loci that may, either alone or in combination with DRD2, enhance prediction of alcoholism, we have found a novel association between a functional coding variant (+118A) within the human mu-opioid receptor gene and alcohol dependency. However, no association was detected between the DRD2 TaqI A1 allele and alcoholism in our sample nor did we find synergy between +118A and TaqI A1 alleles on prediction of risk for the disease. These results suggest that, at the molecular level, the endogenous mu-opioid receptor system is a contributing factor to the etiology of alcoholism.

Alzheimers disease is not associated with the hypertension genetic risk factors PLA(2) or G protein beta3, either independently or interactively with apolipoprotein E.

Growing evidence suggests that hypertension and Alzheimers disease (AD) may share a common etiology. To evaluate the contribution to AD of genetic factors associated with hypertension, we genotyped clinic and community-based AD cases and controls for polymorphisms within the pancreatic PLA(2) gene and the G protein beta3 subunit gene, both of which are located on chromosome 12. Our results do not support an independent association between either of these genes and AD. We further assessed the possibility that either of these genes may interact with the apolipoprotein E gene, a known risk factor for hypertension and AD, on predicting AD. We were unable to find statistical interaction between either the pancreatic PLA(2) or Gbeta3 genes and the apolipoprotein E gene on risk for AD. These results do not support a shared genetic etiology between hypertension and AD. Possibly, a clinical association between these diseases could be due to pathophysiologic interactions.

CD40 signaling and Alzheimer's disease pathogenesis.

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40-CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor-alpha secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-beta peptides. Data reviewed herein show that CD40 ligand and amyloid-beta act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1 beta by endothelial cells and tumor necrosis factor-alpha by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-beta initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.

A beta vasoactivity: an inflammatory reaction.

Mounting evidence from in vitro and in vivo studies in transgenic mice overproducing beta-amyloid peptides (A beta) suggests that A beta can induce vasoconstriction and decrease cerebral blood flow. In this report, we describe the vasoactive properties of A beta, in particular the enhancement of endothelin-1-induced vasoconstriction and A beta's induction of a long-lasting vasoconstrictive event. Furthermore, we show that low doses (as low as 50 nM) of freshly solubilized A beta similar to those observed in the plasma of patients suffering from Alzheimer's disease are vasoactive. By using various inhibitors and activators of the phospholipase A2 (PLA2)/arachidonic acid (AA) cascade, we demonstrate that A beta vasoactivity is dependent on activation of this intracellular signaling pathway, resulting in stimulation of downstream cyclooxygenase-2 and 5-lipoxygenase, which mediate production of proinflammatory eicosanoids. Taken together, our data show that A beta directly activates an intracellular proinflammatory pathway, which is responsible for its vasoactive properties.

The opioid receptor system and alcoholism: a genetic perspective.

Over the past decade, mounting evidence has implicated the endogenous opioid receptor system as a central player in the etiology of alcohol drinking behavior in animals and alcoholism in humans. Much of this work is a product of a pharmacological approach, where differences in opioid receptor pharmacology have been found to predict drinking behavior in animal models of alcoholism, including rats and mice selectively bred for alcohol preference and avoidance. This review considers the opioid receptor system and alcoholism from a genetic standpoint, and discusses investigation into opioid receptor pharmacology in animal models of alcoholism as work that paved the way for the more recent molecular genetic studies implicating the delta-, and particularly, the mu opioid receptors as genetically linked to alcoholism-associated phenotypes in animal models of the disease. These genetic studies are set within the broader context of the candidate gene approach for alcoholism, where opioid receptor genes are taken to be partial, rather than complete, risk factors for alcoholism. Building upon these findings, the recent genetic association between alcoholism and the mu opioid receptor gene in humans is discussed. Finally, the translation of such genetic association studies between opioid receptor genes and alcoholism to a pharmacogenetic approach, allowing for the evaluation of putative relationships between genotype and pharmacological response profiles, is suggested to address the etiological question of what the molecular mechanism is underlying opioid receptor genetic risk for alcoholism phenotypes.

Novel strategies for opposing murine microglial activation.

Pathologic microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially neurotoxic agents, such as pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-alpha). Using cultured N9 microglial cells, we have examined the regulation of TNF-alpha following endotoxic insult with lipopolysacharide (LPS), focusing on the role of the pro-inflammatory phospholipase A2/mitogen activated protein kinase/arachidonic acid/cyclo-oxygenase-2 cascade and the nitric oxide/cGMP pathway. Data show that various inhibitors of the PLA2 cascade markedly inhibit LPS-induced TNF-alpha release, supporting a key role of this pathway in the regulation of microglial activation. We also investigated the putative effects of cGMP-elevating agents on blocking microglial activation induced by LPS. Data show that each member of this class of cGMP-elevating compounds that we employed opposed microglial TNF-alpha release, suggesting that strengthening intracellular cGMP signaling mitigates against microglial activation. Taken together, our results suggest novel strategies for reducing microglial activation.

Isoform-specific vasoconstriction induced by apolipoprotein E and modulation of this effect by Alzheimer's beta-amyloid peptide.

Abeta peptides are thought to be centrally involved in Alzheimer's disease (AD) pathogenesis, although Abeta's pathophysiological mechanisms remain to be elucidated. We previously showed that soluble beta-amyloid1-40 (Abeta) and Abeta1-42 exhibit vasoactive properties, and are able to promote vasoconstriction in rat aortae induced by an endogenous vasoconstrictor, endothelin-1. It is well established that the APOE epsilon4 allele confers risk for both familial and sporadic AD, as well as for hypertension. We now report that physiologic amounts (10 nM) of specific human recombinant apoE isoforms are vasoactive (E4 > E3, and not E2) in isolated rat aortae. In order to investigate if various apoE isoforms could modulate Abeta vasoactivity, we co-incubated Abeta1-40 with various isoforms of apoE in our tissue bath system. Our results show that, while none of the APOE isoforms are able to affect the maximum constriction induced by Abeta; the apoE E4 isoform synergistically enhances the rate of vasoconstriction induced by Abeta. Our data suggest that apoE may promote hypertension and contribute to AD pathogenesis via enhancement of vasoconstriction, and support a link between hypertension, cerebral amyloid angiopathy and AD.

The -491A/T apolipoprotein E promoter polymorphism association with Alzheimer's disease: independent risk and linkage disequilibrium with the known APOE polymorphism.

The epsilon4 allele of the apolipoprotein E gene (APOE) has repeatedly been associated with increased risk for Alzheimer's disease (AD). Bullido and colleagues recently identified a polymorphism in the promoter region of the APOE gene (-491A/T) and found that -491A homozygosity predicted AD independently of APOE epsilon4. Since the -491A/T polymorphism and the known APOE polymorphism must be in tight linkage disequilibrium, and the later polymorphism is know to be associated with the disease, we wished to determine to what extent this linkage disequilibrium explained the -491A/T polymorphism association with Alzheimer's disease. We genotyped a community-based control sample (n = 132) and a clinic-based Alzheimer's disease sample (n = 190) for the known APOE and -491A/T polymorphisms, and find that, while the -491A/T polymorphism confers some independent risk for AD, linkage disequilibrium between the known APOE and -491A/T polymorphic sites explains most of the -491A association. Furthermore, when considering the known APOE and -491A/T polymorphisms alone, APOE epsilon4 status is the best predictor of the disease.

No genetic association between polymorphisms in the Tau gene and Alzheimer's disease in clinic or population based samples.

Mutations in the tau protein gene have recently been found to cause familial fronto-temporal dementia in a number of kindreds demonstrating linkage to chromosome 17. Given that tau pathology is a hallmark of Alzheimer's disease (AD), this raises the possibility that mutations in tau may also be associated with AD. We have investigated the allelic frequencies of polymorphisms in the Tau gene for a possible allelic distortion in Alzheimer's cases, which might suggest a conferred genetic risk. We have genotyped 65 community-based and 200 clinic-based AD cases, and 142 community-based controls at the Tau exon 6 AflIII and BslI polymorphisms and find no independent association with risk for AD in these samples. Further analysis including APOE genotypes from the same samples demonstrated no interaction between either of these polymorphisms and APOE in conferring risk for AD. In addition, haplotype analysis across both sites revealed no difference in haplotype frequencies between cases and controls, nor any interaction with APOE. Therefore our data do not suggest any association between these variations in the Tau gene and Alzheimer's disease.

The alpha-2 macroglobulin gene is not associated with Alzheimer's disease in a case-control sample.

Genetic association has recently been reported between alleles in the alpha-2-macroglobulin (A2M) gene and the occurrence of Alzheimer's disease (AD) in familial and sporadic samples. We have investigated the A2M intronic deletion polymorphism in a case-control study of 295 unrelated clinic and community-based AD cases, and compared these to a sample of 113 unrelated control individuals recruited as part of an epidemiological study. Our results show no association between A2M and AD in either case sample. Furthermore, A2M is not predictive of AD in an interactive fashion when considering APOE, race or gender. In a subset of our larger sample we have also investigated the A2M Val1000lle polymorphism, and again find no evidence for association. We conclude that there is no genetic association between A2M and AD in our case-control sample.

Characterization of murine immunoglobulin G antibodies against human amyloid-beta1-42.

It has been demonstrated that immunization of transgenic mouse models of Alzheimer's disease (AD) with amyloid-beta1-42 peptide (Abeta1-42) results in amelioration of AD-like pathology, including reduced soluble and deposited beta-amyloid and decreased cognitive impairment. Based on the proposed importance of immunoglobulin G (IgG) anti-Abeta antibodies (Abs) in these effects, we sought to characterize these Abs in splenocytes from mice immunized with Abeta1-42. Data show that a more aggregated preparation of Abeta1-42 gives a robust IgG anti-Abeta Ab response, while these Abs are almost undetectable when a less aggregated preparation of Abeta1-42 is used as the immunogen. Importantly, IgG anti-Abeta Ab production is detected after just 12 weeks of Abeta1-42 treatment. Analysis of anti-Abeta Ab IgG isotypes reveals that the majority of these Abs are IgG1, with significantly fewer Abs of the IgG2a or IgG2b isotypes (IgG1>IgG2a>IgG2b), suggesting a T lymphocyte helper type II response after Abeta1-42 immunization. To determine the epitope of Abeta recognized by IgG anti-Abeta Abs, intact Abeta and Abeta peptide fragments were analyzed for their ability to bind these Abs. Data show that these Abs specifically recognize an amino-terminal epitope of Abeta between amino acids one and twelve, with higher affinity for a more soluble preparation of Abeta1-42. These data further indicate the immunogenic potential of Abeta1-42 and offer insight into the nature of the IgG anti-Abeta Ab response.

Bcl-X(L) inhibits apoptosis and necrosis produced by Alzheimer's beta-amyloid1-40 peptide in PC12 cells.

Recent studies have shown that neuronal apoptosis induced by the Alzheimer's disease (AD) beta-amyloid peptide (Abeta) is related to alteration of the Bax/Bcl-2 ratio. It has been demonstrated that Bcl-X(L) (Bcl-X(L) = protein, bcl-X(L) = gene), a Bcl-2-related protein, prevents apoptosis in mammalian cells. Additionally, TGF-beta1 is able to protect cultured neuronal cells from Abeta-induced apoptosis via upregulation of bcl-X(L) and bcl-2 gene expression. We show that Abeta treatment (500 nM, freshly solubilized) results in apoptosis and necrosis in differentiated PC12 cells maintained with a low dose of NGF-beta (1 ng/ml). To investigate whether transfection of PC12 cells with bcl-X(L) could block Abeta-induced apoptosis, we transfected these cells with a bcl-X(L) construct (pcDNA-bcl-X(L)). Data show that bcl-X(L) significantly inhibits both early-stage apoptosis and late-stage apoptosis/necrosis produced by Abeta treatment (1000 nM) in pcDNA3-bcl-X(L)-transfected PC12 cells as compared with pcDNA3 vector-transfected PC12 cells. These results suggest that Bcl-X(L) exhibits both anti-necrotic as well as anti-apoptotic roles in Abeta-challenged PC12 cells.

No association between the low density lipoprotein receptor-related protein (LRP) gene and late-onset Alzheimer's disease in a community-based sample.

It is now commonly known that possession of the epsilon4 allele of the apolipoprotein E (APOE) gene confers an increased risk for both familial and sporadic Alzheimer's disease (AD), in a dose-dependent way. Other genes that may play a role in AD, either through independent association with the disease or through modification of the existing APOE risk, have been reported with conflicting results. One such gene, the low density lipoprotein receptor-related protein (LRP) gene, was recently reported by two groups to be associated with AD, although the groups identified different risk-conferring alleles. Both studies were based on clinic-derived AD populations (one American, one French), and both reported only marginally significant results. We have genotyped a community-based AD and control population at this LRP polymorphism and find no association between the variants at that polymorphism and the occurrence of AD. Further, despite the biochemical relationship between LRP and the ApoE protein, we find no significant statistical interaction between the alleles at these loci.